Ultraviolet-activated conjugation systems and methods

Abstract

Some aspects of the present disclosure are generally related to systems and methods of facilitating conjugation between polymers and molecules of interest, for instance, between a conductive polymer and a biomolecule for biosensing applications. For example, in some embodiments, a UV-activated molecule is used to modify a polymer such that the polymer includes a leaving group, whereafter the leaving group of the polymer may facilitate binding of the polymer to a biomolecule of interest. In some cases, a conductive polymer and sulfo-SANPAH may be exposed to UV light such that at least a portion of the sulfo-SANPAH binds to the polymer. The sulfo-SANPAH may facilitate binding of a biomolecule to the conductive polymer. Still other aspects are generally directed to related articles, systems, kits, methods of use, or the like.

Description

[0001] ULTRAVIOLET-ACTIVATED CONJUGATION SYSTEMS AND METHODS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 727,891, filed December 4, 2024, entitled “Ultraviolet- Activated Conjugation Systems and Methods,” by Ingber, et al., incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] Ultraviolet (UV)-activated conjugation systems and methods are generally described.

[0006] BACKGROUND

[0007] The emergence of point-of-care (POC) diagnostics is reshaping healthcare by facilitating rapid, decentralized, and personalized biomarker detection. Among these technologies, electrochemical biosensors offer unique advantages due to their inherent portability, cost-effectiveness, and real-time analysis capabilities. However, the widespread adoption of electrochemical biosensors is hindered by limitations in surface chemistries for biomolecule immobilization. Typical surface chemistries often implement multi-step processes. Surface chemistries leveraging polymers often include polymers which may not contain suitable functional groups, requiring further functionalization of the polymer to obtain suitable biosensors. Moreover, some electrode materials such as increasingly popular carbon-based electrodes, such as those comprising graphene, require acidic treatment or other such methods for functionalization, which are not easily scalable. Accordingly, improved systems and methods are needed.

[0008] SUMMARY

[0009] UV-activated conjugation systems and methods are generally described. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0010] Some aspects are related to methods.

[0011] In some embodiments, the method comprises providing a surface comprising a polypyrrole coating; and binding sulfo- SANP AH to the polypyrrole using ultraviolet light. In some embodiments, the method further comprises reacting the sulfo-SANPAH to a biomolecule to bind the biomolecule to the polypyrrole. In some embodiments, the

[0012] #14581396vl method further comprises exposing the surface to ultraviolet light. In some embodiments, the method further comprises binding an antigen using an antibody.In some embodiments, the method further comprises using the surface for biosensing.

[0013] In some embodiments, the method comprises exposing a conductive polymer coated on a surface of an electrode and sulfo-SANPAH to UV light to bind the sulfo- SANPAH to the conductive polymer; and binding a biomolecule to the conductive polymer.

[0014] In some embodiments, the method is for modifying a conductive polymer. In some embodiments, the method comprises exposing a conductive polymer and a UV- activatable molecule to UV light to bind the UV- activatable molecule to the conductive polymer to form a modified conductive polymer; and binding the modified conductive polymer to a biomolecule.

[0015] Some aspects are related to articles.

[0016] In some embodiments, the article comprises an electrode comprising a polypyrrole coating and a biomolecule bound to the polypyrrole coating via an amide linkage. In some embodiments, the article further comprises a voltage source in electrical communication with the electrode.

[0017] In some embodiments, the article is for biosensing. In some embodiments, the article comprises an electrode; a conductive polymer disposed on a surface of the electrode; and a biomolecule covalently bound to the conductive polymer, wherein the conductive polymer comprises a functional group including a nitrophenyl ring.

[0018] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale unless otherwise indicated. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every

[0021] #14581396vl component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0022] FIG. 1A is a schematic diagram of a method of modifying an electrode surface, according to some embodiments;

[0023] FIG. IB is a schematic diagram showing the reaction between a polypyrrole surface, sulfo-SANPAH, and a BSA conjugate, according to some embodiments;

[0024] FIG. 1C is a schematic diagram showing the reaction between an electrode surface, sulfo-SANPAH, and a BSA conjugate, according to some embodiments;

[0025] FIG. 2 is a cyclic voltammogram (CV) of an electropolymerization process, according to some embodiments;

[0026] FIG. 3 is a plot of CVs of various electrodes, according to some embodiments;

[0027] FIG. 4 is a plot of the peak current measured at electrodes fabricated using different conditions, according to some embodiments;

[0028] FIG. 5 is a plot of the peak current measured at electrodes exposed to different conditions, according to some embodiments; and

[0029] FIG. 6 is a plot of the peak current measured at electrodes fabricated using different conditions, according to some embodiments.

[0030] DETAILED DESCRIPTION

[0031] Some aspects of the present disclosure are generally related to systems and methods of facilitating conjugation between polymers and molecules of interest, for instance, between a conductive polymer and a biomolecule for biosensing applications. For example, in some embodiments, a UV-activated molecule is used to modify a polymer such that the polymer includes a leaving group, whereafter the leaving group of the polymer may facilitate binding of the polymer to a biomolecule of interest. In some cases, a conductive polymer and sulfo-SANPAH may be exposed to UV light such that at least a portion of the sulfo-SANPAH binds to the polymer. The sulfo-SANPAH may facilitate binding of a biomolecule to the conductive polymer. Still other aspects are generally directed to related articles, systems, kits, methods of use, and the like.

[0032] Biological sensors, or biosensors, are often configured to detect the presence and / or quantify the concentration of a target molecule in clinical and / or environmental settings. Typically, biosensors leverage capture molecules configured to bind to the

[0033] #14581396vl target molecule, for instance, a capture molecule comprising antibody configured to bind a target molecule comprising an antigen, to detect and / or sense the presence of the biomolecule. However, conventional methods for introducing the molecules configured to bind to the target molecule (e.g., the antibody) into a biosensor are not simple, often requiring lengthy and / or complex synthetic methods. Thus, many conventional biosensors are not scalable for commercial settings.

[0034] Accordingly, some aspects of the present disclosure are generally related to improved systems and methods of modifying surfaces such that they are functionalized for use in biosensors. Some embodiments may utilize polymers, for instance, conductive polymers, as platforms for biosensors, due to the variety of available chemistries and relatively simple incorporation of such polymers into typical platforms. Using polymers, however, is complicated due to polymers often lacking functional groups that are easily conjugated to the molecule configured to bind to the biomolecule of interest (e.g., an antibody). For instance, conventional methods for modifying polymers may utilize thiol chemistry and / or EDC / NHS chemistry to include functional groups in the monomers, prior to polymerization. These additional steps may be limiting, complex, lengthy, and / or expensive. For example, thiol chemistry is limited to binding to metallic surfaces such as gold, mercury, platinum, and / or cadmium. Similarly, EDC / NHS chemistry is limited to modifying polymers having a COOH group, and the chemistry is highly sensitive to the pH and temperature conditions during the reactions. Moreover, these commonly used thiol chemistry and / or EDC / NHS chemistry may lead to undesirable internal crosslinking of the polymer, e.g., rather than leaving the functional groups readily available for subsequent reactions.

[0035] Advantageously, some embodiments described herein are related to using UV- activatable molecules to functionalize polymers for biosensors. For example, consider FIG. 1A, which is a non-limiting schematic illustration of an example method of modifying a surface with a biomolecule using a UV-activated molecule 100. An electrode surface 105 is contacted with a solution containing monomeric pyrrole 110, and the electrode is electrochemically cycled 115 to polymerize the pyrrole and form a polypyrrole coating 120 on the electrode surface 105. The polypyrrole coating 120 is then exposed to UV light in the presence of a solution containing sulfo-SANPAH 125 such that the polypyrrole comprises at least a portion of the sulfo-SANPAH 130. The

[0036] #14581396vl electrode comprising the at least a portion of the sulfo-SANPAH is then incubated 135 with anti-macrophage inflammatory protein- la antibody (anti-MIP IgG) 136 to form the modified electrode surface 140 comprising the polypyrrole coating 120 and the anti-MIP IgG 136. The remainder of the electrode surface (e.g., the portion where there was not anti-MIP IgG present) was then blocked with bovine serum albumin (BSA) 145 to prevent non-specific binding of the antigen of interest, whereafter a solution containing the MIP antigen (MIP Ag) was exposed 150 to the modified surface 140 to form an antigen-antibody conjugate 155 on the electrode surface.

[0037] Some aspects are directed to articles. In some embodiments, the article comprises a surface comprising a polymer. In some embodiments, the article comprises a surface upon which a polymer is disposed. In some embodiments, the surface comprises an electrode comprising a conductive polymer. In some embodiments, a conductive polymer may be disposed on the electrode. In some embodiments, the article comprises a nanoparticle on which a polymer is disposed. For example, in some embodiments, the article comprises a nanoparticle comprising a conductive polymer disposed thereon.

[0038] Any of a variety of surfaces are suitable for use in an article, according to some embodiments. In some embodiments, the surface is conductive such that it may function as an electrode to electropolymerize a conductive polymer thereon, as described in more detail elsewhere herein. Non-limiting examples of suitable surfaces, for example for use as an electrode, include stainless steel, gold, platinum, glassy carbon, and screen printed carbon electrodes. Other surfaces are also possible.

[0039] The article may comprise any of a variety of suitable polymers. In some embodiments, the polymer is conductive. In some embodiments, the polymer comprises polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA), polycarbonate (PC), cyclic olefin copolymer (CoC), organic fibre-based polymers (e.g., cellulose, chitin, agarose, etc.), polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene) (PEDOT), polyethylene (PE), polyvinyl chloride (PVC), polyacrylamide, and / or polyhydroxyethylmethacrylate (pHEMA). In some embodiments, the monomeric units of the polymer and / or the polymer comprise secondary amines. For instance, in some embodiments, the polymer comprises polypyrrole and / or polyaniline. As described above, the polymer may be disposed on the surface of the article. In some embodiments, the surface is an electrode surface, and the polymer is disposed thereon. In some

[0040] #14581396vl embodiments, the polymer is present as a coating such that the article comprises a surface comprising a polymer coating. The polymer coating, in accordance with some embodiments, may be present on at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, up to 100% of the surface area of the surface (e.g., the electrode surface).

[0041] The polymer coating may further comprise any of a variety of suitable nanomaterials, in accordance with some embodiments. In some embodiments, nanomaterial additives to the polymer coating may alter and / or enhance various properties (e.g., conductivity) of the polymer coating. In some embodiments, the polymer coating may comprise nanomaterials comprising carbon (e.g., reduced graphene oxide (rGO), carbon nanotubes), gold (e.g., gold nanoparticles, gold nanocrystals), and / or zinc oxide (ZnO).

[0042] The polymer coating may have any suitable thickness, in accordance with some embodiments. For instance, in some embodiments, an average thickness of the polymer coating is greater than or equal to 1 micron, greater than or equal to 10 microns, greater than or equal to 100 microns, greater than or equal to 500 microns, greater than or equal to 1 mm, or greater than or equal to 1.5 mm. In some embodiments, the average thickness of the polymer coating is less than or equal to 2 mm, less than or equal to 1.5 mm, less than or equal to 1 mm, less than or equal to 500 microns, less than or equal to 100 microns, or less than or equal to 10 microns. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 10 microns and less than or equal to 2 mm). Other ranges are also possible.

[0043] The polymer coating may be substantially uniform in accordance with some embodiments. For instance, in some embodiments, a thickness of the polymer coating of the surface (e.g., the electrode surface) may deviate by no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% from an average thickness.

[0044] The polymer coating may be present on any suitable amount of the surface (e.g., the electrode surface), in accordance with some embodiments. In some embodiments, the polymer coating may be present on at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, and / or up to 100% of the surface.

[0045] #14581396vl - 1 -

[0046] In some embodiments, the polymer may comprise a functional group. In some embodiments, the functional group includes a nitrophenyl ring (I).

[0047] As described elsewhere herein in more detail, the polymer may be modified such that it includes the functional group comprising the nitrophenyl ring.

[0048] In some embodiments, the article further comprises a biomolecule bound to the polymer (e.g., conductive polymer). In some embodiments, the biomolecule is bound to the polymer via a functional group, e.g., comprising a nitrophenyl ring. In some embodiments, the biomolecule comprises an antibody, a protein, an aptamer, a nucleic acid, a peptide, an antigen-carrier protein conjugate, a protein receptor, or the like. For instance, in some embodiments, the biomolecule bound to the polymer is an antibody, and the antibody is bound to the polymer of the article. In some such instances, the biomolecule may be configured to capture or bind to a target molecule, e.g., of the biosensor for which the article is designed. In some embodiments, the biomolecule is chemically bound to the polymer of the article. Any of a variety of suitable chemical bonds may bind the biomolecule configured to bind to a biomolecule of interest to the polymer. In some embodiments, the biomolecule is bound to the polymer coating via an amide linkage. In some embodiments, in place of the biomolecule, a cell may be bound to the polymer coating of the surface.

[0049] In some embodiments, the article is configured for biosensing of a target molecule. For example, in some embodiments, the article comprises an electrode with a conductive polymer disposed thereon, where the conductive polymer comprises a biomolecule configured to specifically bind to the target molecule. As a non-limiting example, the biomolecule bound the polymer coating is an antibody, and the target molecule is an antigen of the antibody.

[0050] In some embodiments, the article may further include a power source (e.g., a power source, a battery, a potentiostat, etc.) associated with the surface and / or the polymer. In some embodiments, the power source is configured to apply a potential

[0051] #14581396vl and / or current to the surface and / or polymer. In some embodiments, the power source is in electrical communication with the surface and / or the polymer. It will be understood that the power source may include at least two electrical connections thereto (e.g., a positive terminal and a negative terminal, an electrical connection for a reference electrode, etc.), where at least one of the electrical connections is configured to establish electrical connection between the power source and the electrode and / or polymer. In some embodiments, a second electrical connection may connect to a second electrical connection, e.g., a counter electrode. There may be an electrolyte solution present between the first and second electrical connections, e.g., to establish a circuit through which current may flow. Other arrangements of the power source relative to the electrode and / or the polymer are possible.

[0052] Some aspects are generally related to systems. In some embodiments, the system includes the article. In some embodiments, the system is configured to detect and / or determine a concentration of a target molecule. For example, in some embodiments, a system may include an article as described herein and a sample solution containing a target molecule. In some embodiments, the system is configured to make the article. Accordingly, in some embodiments, the system may include a UV light source configured to emit UV light at at least a portion of a polymer coating on a surface of the article.

[0053] Some aspects are generally related to methods. In some embodiments, methods of making the articles and / or systems disclosed herein are generally described.

[0054] In some embodiments, the method comprises providing a surface. A described elsewhere herein, any of a variety of surfaces are suitable, in accordance with some embodiments. A surface may include an exposed portion of an article comprising the portion, in some embodiments, such that the surface may interact with a solution and / or be modified. In some cases, the surface includes exposed portions that are conductive, e.g., such that the surface may be modified using electrochemical techniques as described elsewhere herein. In some embodiments, the method includes providing a surface comprising a polymer. For instance. In some embodiments, the method includes providing a surface comprising a polymer coating. In some embodiments, the method includes providing a surface comprising a polypyrrole polymer coating.

[0055] #14581396vl In some embodiments, when the surface is provided, the surface may not comprise a polymer coating. In some such embodiments, the method may include polymerizing a polymer onto the surface, e.g., to form a coating. Conventional polymerization methods may be used in accordance with some embodiments. For example, the method may include providing a solution comprising monomers of the desired polymer, and contacting the surface with the solution. In some embodiments, the method may include heating the solution and / or the surface, exposing the solution and / or the surface to UV light, or the like. In some embodiments when the surface is an electrode surface, the method comprises electropolymerizing a polymer coating on the electrode surface. Any of a variety of suitable electrochemical techniques are possible for electropolymerizing a polymer onto a surface, in accordance with some embodiments. For example, in some embodiments, electropolymerization may occur under constant current conditions. In some embodiments, electropolymerization may occur under constant potential conditions. In some embodiments, a potential sweep method may be used, e.g., cyclic voltammetry for electropolymerization.

[0056] The method may further comprise binding at least a portion of a UV- activatable molecule to the polymer coating on the surface. In some embodiments, UV-activatable means that, in the presence of certain wavelengths of UV light, chemical reactions between the UV activatable molecule and the polymer coating occur at a faster rate than in the absence of the UV light. In some embodiments, the rate of reaction between the UV activatable molecule and polymer coating is substantially faster in the presence of the UV light compared to when the UV light is absent. In some embodiments, the rate of reaction between the UV activatable molecule and polymer coating is at least 1.5 times, at least 2 times, at least 5 times, at least 100 times, or at least 1,000 times faster in the presence of the UV light compared to when the UV light is absent. In some embodiments, the reaction may not measurably proceed in the absence of UV light. Suitable UV activatable molecules include at least a portion that is activatable in the presence of UV light. In some embodiments, the UV-activated molecule is sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH).

[0057] In some embodiments, the method includes exposing the polymer coating and the UV-activatable molecule to UV light to bind at least a portion of the UV-activatable molecule to the conductive polymer. In some embodiments, to facilitate binding

[0058] #14581396vl therebetween, the polymer coating and the UV- activatable molecule may be exposed to UV light to bind at least a portion of the UV-activatable molecule to the polymer coating, thereby forming a modified polymer coating. In some embodiments, the polymer coating is a conductive polymer. In some such embodiments, the conductive polymer and the UV-activatable molecule may be exposed to UV light to bind at least a portion of the UV-activatable molecule to the conductive polymer, thereby forming a modified conductive polymer. In some embodiments, the UV-activatable molecule comprises sulfo-SANPAH and the polymer coating comprises polypyrrole. In some such embodiments, the sulfo-SANPAH and the polypyrrole coating are exposed to UV light to bind at least a portion of the sulfo-SANPAH to the polypyrrole to form a modified polypyrrole. In some embodiments, at least the N-hydroxysulfosuccinimide portion of the sulfo-SANPAH may bind to the polymer coating. It will be understood that the UV- activatable molecule (e.g., sulfo-SANPAH) may be present in a solution, and the solution may be in contact with the polymer coating present on the surface.

[0059] Exposing the polymer coating and the UV-activatable molecule to UV light may proceed by any of a variety of suitable methods. In some embodiments, a UV lamp may be used. In some embodiments, exposure to UV light from a common overhead light or ambient source (e.g., sunlight) is also possible. In some embodiments, a laser configured to emit UV light may be used. It may be desirable to use a laser to direct the UV light to a certain portion of the solution containing the UV activatable molecule, for instance, the polymer coating on the surface to which at least a portion of the UV activatable molecule is to be bound. In some embodiments, prior to exposing the polymer coating and the UV- activatable molecule to UV light, the solution containing the polymer coating may not be exposed to substantial UV light, so as to keep the UV activatable molecule inactive (e.g., unreacted). Avoiding exposure to UV light include keeping the UV activatable molecule in darkness and / or under a light source that does not emit UV light.

[0060] In some embodiments, the UV light source may emit light having any of a variety of suitable wavelengths. In some embodiments, the UV light source may emit light having a wavelength of greater than or equal to 10 nanometers, greater than or equal to 50 nanometers, greater than or equal to 100 nanometers, greater than or equal to 200 nanometers, greater than or equal to 250 nanometers, greater than or equal to 280 nanometers, greater than or equal to 300 nanometers, greater than or equal to 320

[0061] #14581396vl nanometers, or greater than or equal to 350 nanometers. In some embodiments, the UV light source may emit light having a wavelength of less than or equal to 400 nanometers, less than or equal to 350 nanometers, less than or equal to 320 nanometers, less than or equal to 300 nanometers, less than or equal to 280 nanometers, less than or equal to 250 nanometers, less than or equal to 200 nanometers, less than or equal to 100 nanometers, or less than or equal to 50 nanometers. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 10 nanometers and less than or equal to 400 nanometers). Other ranges are also possible. In some embodiments, the UV light source may be selected based on the wavelength(s) of light that activate the UV activatable molecule. For example, in some embodiments, a UV light source may emit light having a wavelength of greater than or equal to 320 nanometers and less than or equal to 350 nanometers when the UV- activatable molecule comprises sulfo-SANPAH.

[0062] In accordance with some embodiments, exposing the polymer coating and the UV-activatable to UV light comprises exposing the polymer coating and the UV- activatable to UV light for at least 10 seconds, at least 1 minute, at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 6 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 6 days. In some embodiments, exposing the polymer coating and the UV-activatable to UV light comprises exposing the polymer coating and the UV-activatable to UV light for no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, no more than 1 day, no more than 12 hours, no more than 6 hours, no more than 1 hour, no more than 45 minutes, no more than 30 minutes, no more than 20 minutes, no more than 10 minutes, no more than 5 minutes, no more than 3 minutes, no more than 2 minutes, or no more than 1 minute. Combinations of the foregoing ranges are possible. Other ranges are also possible.

[0063] Binding at least a portion of the UV-activatable molecule to the polymer coating may advantageously add functional groups from the UV activatable molecule to the polymer coating such that, in some embodiments, further chemical reactions may occur using the functional groups of the polymer coating. This may desirably facilitate further modification of the polymer coating, e.g., at the functional group. As a non-limiting example, in some embodiments, the UV activatable molecule is sulfo-SANPAH, and at

[0064] #14581396vl least the N-hydroxysulfosuccinimide portion of the sulfo-SANPAH binds to the polymer coating such that the N-hydroxysulfosuccinimide may then operate as a leaving group from the polymer coating, e.g., during chemical reactions.

[0065] Accordingly, in some embodiments, the method further comprises reacting the at least a portion of the UV- activatable molecule bound to the polymer coating (e.g., a conductive polymer coating) with a biomolecule to bind the biomolecule to the polymer coating. For instance, in some embodiments, the method includes reacting the at least a portion of the sulfo-SANPAH bound to a polypyrrole coating to a biomolecule to bind the biomolecule to the polypyrrole. The biomolecule may be any suitable biomolecule as described elsewhere herein. For example, in some embodiments the biomolecule is an antibody, and the method includes reacting the at least a portion of the sulfo-SANPAH bound to a polypyrrole coating to the antibody to bind the antibody to the polypyrrole. It will be understood that the biomolecule (e.g., an antibody) may be present in a solution, and the solution may be in contact with the polymer coating present on the surface (e.g., the conductive polymer coating on the electrode surface).

[0066] For example, FIG. IB is a schematic diagram showing the chemical reaction between sulfo-SANPAH 160 and a polypyrrole (Ppy) surface 162. In this reaction, the sulfo-SANPAH 160 is exposed to UV light 162 to activate the nitophenyl azide of the sulfo-SANPAH to form activated sulfo-SANPAH 164, where the activated sulfo- SANPAH 164 reacts with a polypyrrole surface 166 to form a modified polypyrrole 168 comprising a portion of the sulfo-SANPAH including the nitrophenyl ring. The modified polypyrrole 168 may be exposed to a BSA conjugate 169 to bind the BSA to the polypyrrole 170. FIG. 1C shows a similar example to FIG. IB, but in this case more general than FIG. IB. For example, sulfo-SANPAH 171 and an electrode 172 with a polymer coating 174 is exposed to UV light 176 to form a modified polymer coating 178. The modified polymer coating 178 may then be exposed to a biomolecule 179 to bind the biomolecule to the polymer coating 180.

[0067] It will be understood that certain methods may further include one or more washing steps, optionally between any other method steps described herein, according to some embodiments. Non limiting examples of a washing solution, e.g., which may be used in a washing step, may comprise deionized water (e.g., having a resistivity of greater than or equal to 15 megaOhms, greater than or equal to 16 megaOhms, greater

[0068] #14581396vl than or equal to 17 megaOhms, greater than or equal to 18 megaOhms, or greater than or equal to 18.2 megaOhms) or phosphate buffer solution (PBS). Other washing solutions are also possible. In some cases, the method many further include a blocking step, wherein a molecule such as BSA is incubated with the surface (e.g., electrode surface) to fill in any portion of the surface that is otherwise free of species (e.g., free of polymer, biomolecule, etc.), the blocking step, in some embodiments, may prevent non-specific adsorption of a target molecule of the biosensor, e.g., when using the biosensor.

[0069] Advantageously, in some embodiments, the methods described herein may facilitate high throughput modification of a plurality of surfaces (e.g., electrode surfaces). For example, a plurality of surfaces may be modified in parallel. For example, in some embodiments, the plurality of surfaces is a plurality of electrode surfaces. In some embodiments, the plurality of electrode surfaces includes an electrode surface of a first electrode, an electrode surface of a second electrode, an electrode surface of a third electrode surface, and so forth. In some embodiments, the plurality of electrode surfaces may be positioned within a bath containing a solution containing a monomer, and a multiplexed potentiostat may be configured to polymerize the monomer into a polymer on each of the electrode surfaces of the plurality of electrode surfaces. In some embodiments, the plurality of electrode surfaces (e.g., optionally with a respective polymer coating on each of the electrode surfaces) may be positioned within a bath containing a solution containing a UV-activatable molecule. Accordingly, the polymer coating of each electrode surface may individually or simultaneously be exposed to UV light while in contact with the solution containing the UV-activatable molecule to simultaneously bind at least a portion of the UV-activatable molecule to each of the polymer coatings of the plurality of electrode surfaces. The solution contained within the bath may then be refreshed with another solution containing a biomolecule, whereafter the biomolecule may bind to the at least a portion of the UV-activatable molecule bound to each of the polymer coatings of the plurality of electrode surfaces.

[0070] In some embodiments, methods of using the articles and / or systems described herein are generally described. In some embodiments, the articles and / or systems may include a biomolecule configured to selectively bind to a target molecule. For instance, the biomolecule of the article and / or system may comprise an antibody configured to bind to a target antigen. In some embodiments, the articles and / or systems may be

[0071] #14581396vl configured to bind to a target molecule, e.g., for biosensing applications. Accordingly, some aspects are related to methods of using the articles and / or systems described herein for biosensing applications. In some embodiments, the biosensing application is for monitoring a biomarker or other molecule of interest from a biological sample (e.g., blood, bodily fluid, etc. form a subject), for instance, in a clinical setting. In some embodiments, the biosensing application is for monitoring a biomarker or other molecule of interest from an environmental sample.

[0072] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0073] EXAMPLE 1

[0074] This example generally describes the fabrication of an article as described herein.

[0075] There exist many different techniques and approaches, commonly referred to as surface chemistries, to modify the surface of electrodes for the purpose of developing biological sensors, commonly termed “biosensors,” for the detection of target molecules in clinical and environmental applications. The majority of existing techniques require approaches that are not easily scaled in commercial settings and / or require lengthy or complex synthesis processes. One example is related to polymers without free functional groups like pyrrole. In such cases, chemical modification of monomers are designed and synthesized to incorporate functional groups before polymerization of the monomers, which is a time consuming and costly process. In contrast, in this example, the development of a simple and rapid surface chemistry that integrates rapid electropolymerization of a polymer with a UV-activated linker for simple amide bond conjugation between the linker and probe biomolecules to produce a fully functionalized biosensor platform is described. This rapid, simple, and low-cost technique is desirable when transferring to commercial fabrication methods (e.g., scaling up), as the platform can be readily performed with a multiplexed potentiostat device to polymerized electrodes, batch UV-treating the electrodes immersed in the UV-activated linker, and immersion of the electrodes in a solution containing the probe biomolecule. Furthermore, the first two steps of the surface chemistry (e.g., polymerization and UV-activated linkage) are flexible and may be used with any of a variety of suitable biomolecule probes in various embodiments, thereby facilitating multiplexed functionalization of

[0076] #14581396vl devices for multi-target analysis in the cases of complex sample analysis and / or disease monitoring or prognosis.

[0077] Background

[0078] The emergence of point-of-care (POC) diagnostics is reshaping healthcare by facilitating rapid, decentralized, and personalized biomarker detection. Among these technologies, electrochemical biosensors offer unique advantages due to their inherent portability, cost-effectiveness, and real-time analysis capabilities. However, the widespread adoption of electrochemical biosensors is hindered by limitations in surface chemistries for biomolecule immobilization. Typical surface chemistries often implement multi-step processes. Surface chemistries leveraging polymers often include polymers which may not contain suitable functional groups, requiring further functionalization of the polymer to obtain suitable biosensors. Moreover, some electrode materials such as increasingly popular carbon-based electrodes, such as those comprising graphene, require acidic treatment or other such methods for functionalization, which are not easily scalable. Accordingly, typical methods often rely on complex protocols, expensive materials, or limited target specificity, rendering them unsuitable for high- throughput clinical applications.

[0079] To address these challenges, this example describes a simple, rapid surface chemistries capable of label-free multi-metabolite sensing with high sensitivity and selectivity. This approach additionally eliminates the need for cumbersome labeling or amplification steps, streamlining the analytical process and facilitating the detection of subtle but clinically relevant biomarker variations. The electrochemical biosensors described herein facilitate use of POC diagnostics in early disease detection, personalized medicine, and / or precision healthcare.

[0080] Description

[0081] In this example, the development of a technique for fabricating a simple and rapid surface chemistry for the stable and flexible functionalization of biomolecular probes to facilitate sensitive, selective quantitation of metabolites in complex samples is described. As a case study, pyrrole monomers for forming conductive polypyrrole films were used. FIG. 1A shows a schematic of the example technique described in this Example. Briefly, the techniques involve the electrodeposition of polypyrrole (Ppy), a conductive polymer with plentiful double bonds as well as C-H and N-H site

[0082] #14581396vl nucleophiles, on carbon screen printed electrodes (SPE-Cs). This is followed by rapid UV-assisted binding of a sulfo-SANPAH linker molecule through activation of nitrophenyl azides to form nitrene groups which bind to nucleophiles on the polymer surface. The available sulfo-NHS ester can then react with primary amine groups on the chosen probe biomolecule, in pH 7-9 buffers, resulting in a stable amide bond with the surface and the release of sulfo-N-hydroxy-succinimide.

[0083] The detailed procedure is presented below:

[0084] 1. SPE-C electrodes, kept in a dehumidifier to prevent electrode material oxidation, were cleaned by rinsing with IPA, followed by MilliQ water and dried under a stream of nitrogen.

[0085] 2. Pyrrole was prepared by first diluting in DMSO and then further diluting in MilliQ to a final concentration of 50 mM pyrrole and 1% DMSO then vortexed to mix thoroughly.

[0086] 3. As illustrated in FIG. 1A as step 115, pyrrole was electropolymerized onto the SPE-C working electrode (WE) surface 105 by adding 100 microliters of 50 mM pyrrole and applying the following cyclic voltammetry (CV) procedure shown in a- f. The first and fifth cycle from the CV scans are shown in FIG. 2. a. Start potential: -0.5 V b. Potential vertex 1: 0.8 V c. Potential vertex 2: -0.8 V d. Step potential: 0.01 V e. Scan rate: 0.05 V / s f. Number of cycles: 5

[0087] 4. SPE-Cs were then rinsed with MilliQ water and dried with purified air.

[0088] 5. 4 mg / mL (10 mM) of sulfo-SANPAH was prepared by diluting powder in 50 mM of HEPES buffer (pH 6.5 - 7.0).

[0089] 6. 100 microliters of 4 mg / mL sulfo-SANPAH was then added to the electrode surface and exposed to UV (320-350 nm) for 30 minutes, see step 120 of FIG. 1A.

[0090] 7. Electrodes were then washed with PBS (pH 7.4) and dried with purified air.

[0091] #14581396vl 8. Electrodes modified with Ppy and sulfo-SANPAH were then incubated 100 microliters of various concentration of anti-MIP (macrophage inflammatory protein- la) antibody prepared in 0.1% bovine serum albumin (BSA) in PBS (pH 7.4) for 30 minutes, as shown as step 135 of FIG. 1A.

[0092] 9. Electrodes were washed with PBS and then further incubated with 100 microliters of 2.5% BSA in PBS (pH 7.4) to ensure complete coverage of the electrode surface to prevent non-specific interactions, as shown as step 145 of FIG. 1A.

[0093] 10. Finally, electrodes were washed with PBS (pH 7.4) and PBS (pH 7.4) was added to the electrode surface and stored at 4 degrees Celsius until further use.

[0094] Modified electrodes can then be incubated with various concentrations of MIP antigen prepared in 0.1% BSA in PBS (pH 7.4) for 1 hour and washed with PBS (pH 7.4), as shown as step 150 of FIG. 1A. Electrodes were then measured to determine concentration with a dose response curve. Amperometric measurements were carried out using CV with the following procedure: a. Start potential: -0.3 V b. Potential vertex 1: 0.7 V c. Potential vertex 2: -0.3 V d. Step potential: 0.01 V e. Scan rate: 0.05 V / s f. Number of cycles: 3

[0095] The CV responses for measurement of electrode modification and subsequent binding of probe anti-MIP antibody and antigen (FIG. 3) were recorded in PBS (pH 7.4) containing 5 mM [Fe(CN)6]3’ / 4“ redox couple at scan rates of 50 mV / s. The oxidation and reduction peaks for the bare and Ppy-modified electrodes observed at 0.3 and 0.1 V can be attributed to the presence of the redox molecules in solution and demonstrate the reversibility of the oxidation process from [Fe(CN)e]4’ to [Fe(CN)6]3’. The increase in the current peaks after the electropolymerization of Ppy can be attributed to the increased conductivity of the sensor surface after formation of the pyrrole polymer. The potential of these current peaks shifts to 0.175 and 0.225 V after binding of the sulfo-SANPAH linker which can be explained by the increased electrochemical reversibility of the redox reaction between the electrolyte and the electrode surface. Subsequent decreases in the same current peaks in the step thereafter result from the increased insulating effect of the

[0096] #14581396vl sulfo-SANPAH linker and anti-MIP antibody and antigen binding on the electrode surface.

[0097] Conjugation of the sulfo-SANPAH linker to the conductive Ppy thin-film was carried out by incubating the working electrodes either in the presence or absence of the linker in HEPES buffer while either exposing the surface to UV for 30 minutes or incubating in the dark (FIG. 4). The largest change in the magnitude of current was observed in the case where the linker was present in solution and the electrode was treated with UV light. The next largest observed change in the magnitude of the current was observed when the linker was present in solution but the electrode was not treated in the presence of UV light. The magnitude of the current change in the latter case was less than 50% of the magnitude of the change observed when the electrode was UV treated.

[0098] Conjugation of either M-PEG6-NH2 and anti-MIP IgG were then used to characterize probe functionalization to the sulfo-NHS ester group. Electrodes were incubated with HEPES buffer or sulfo-SANPAH for 30 minutes and exposed to UV or kept in darkness followed by 30 minutes incubation with M-PEG6-NH2 or anti-MIP IgG in 0.1% BSA in PBS. Current was then measured through each of the modified electrodes, results from which are shown in FIG. 5. Conjugation of the anti-MIP IgG gave the highest current peak change with the linker that was UV-activated. However, significant current change was also observed with the other conditions suggesting passive adsorption of the antibodies to the polypyrrole film and / or ionic bonding. When conjugating antibodies as probes for biosensing, it is desirable to ensure that the orientation of the antibodies is correct or else any subsequent signal will lead to a falsepositive through non-specific binding or lack of target binding entirely due to incorrect orientation of the antibody.

[0099] Electrodes at various stages of the process shown in FIG. 1A were additionally cycled in the presence of a redox probe. The (1) electrode, (2) electrode comprising Ppy, (3) the electrode comprising Ppy and the Anti-MIP IgG, (4) the electrode comprising Ppy and the Anti-MIP IgG and blocked with BSA, and (5) the electrode comprising Ppy and the Anti-MIP IgG, blocked with BSA, and incubated with MIP antigen were cycled. The results of the cycling of the electrodes are plotted in FIG. 6. Incubation of 10 ng / mL MIP antigen resulted in a distinct peak current decrease, signifying successful target molecule binding to the fabricated sensor surface-bound antibodies.

[0100] #14581396vl Variations

[0101] • A variety of conductive and non conductive polymers such as polyaniline (PANI), polydopamine (PDA), Poly(3,4-ethylenedioxythiophene) (PEDOT), polyethylene (PE) and polyvinyl chloride (PVC) can be used.

[0102] • The conductive polymer can be further modified with conductive nanomaterials such as reduced graphene oxide (rGO), gold nanoparticles / nanocrystals (AuNPs / AuNCs), zinc oxide (ZnO) or carbon nanotubes (CNTs).

[0103] • The method can also be directly used on carbon or graphene-based electrodes.

[0104] • An array of electrodes functionalized with different biological probe molecules can be implemented for multiplexed detection of metabolites.

[0105] • A variety of redox molecules such as hexacyanoferrate(IVIII), ruthenium(II / III), ferrocene / ferrocenium etc. can be implemented.

[0106] • The screen printed electrodes can be replaced with other types of electrodes such as evaporated metals on glass, printed circuit boards etc. and the carbon can be replaced with other materials such as gold, platinum or indium tin oxide (ITO). Benefits

[0107] Integration of the non-modified pyrrole polymer on carbon screen printed electrodes (SPE-Cs) and the UV-activated amide linker (Sulfo-SANPAH) allows the user to rapidly fabricate a straightforward highly conductive surface chemistry that facilitates reproducible and effective functionalization of a surface (e.g., an electrode surface) with a wide range of biological probe molecules including but not limited to; antibodies, proteins, aptamers, nucleic acids, cells, etc. This further allows sensitive and selective detection of metabolites in complex biological fluids with high levels of stability due to the simplicity of the structure of the proposed surface chemistry.

[0108] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual

[0109] #14581396vl parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0110] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0111] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0112] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general,

[0113] #14581396vl the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0114] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0115] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

[0116] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a

[0117] #14581396vl certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0118] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0119] #14581396vl

Claims

CLAIMSWhat is claimed is:

1. A method, comprising: providing a surface comprising a polypyrrole coating; and binding sulfo- SANP AH to the polypyrrole using ultraviolet light.

2. The method of claim 1, further comprising reacting the sulfo-SANPAH to a biomolecule to bind the biomolecule to the polypyrrole.

3. The method of claim 1 or 2, wherein the surface is an electrode surface.

4. The method of claim 3, further comprising a voltage source in electrical communication with the electrode.

5. The method of any one of the preceding claims, further comprising exposing the surface to ultraviolet light.

6. The method of any one of claims 2-5, wherein the biomolecule is an antibody.

7. The method of claim 6, further comprising binding an antigen using the antibody.

8. The method of any one of the preceding claims, further comprising using the surface for biosensing.

9. An article, comprising: an electrode comprising a polypyrrole coating and a biomolecule bound to the polypyrrole coating via an amide linkage.

10. The article of claim 8, further comprising a voltage source in electrical communication with the electrode.#14581396vl11. A method, comprising : exposing a conductive polymer coated on a surface of an electrode and sulfo- SANPAH to UV light to bind the sulfo- SANP AH to the conductive polymer; and binding a biomolecule to the conductive polymer.

12. A method of modifying a conductive polymer, comprising: exposing a conductive polymer and a UV-activatable molecule to UV light to bind the UV-activatable molecule to the conductive polymer to form a modified conductive polymer; and binding the modified conductive polymer to a biomolecule.

13. The method of claim 12, wherein the conductive polymer is present as a coating on an electrode surface14. The method of claim 12 or 13, wherein the UV-activatable molecule is sulfo- SANPAH.

15. The method of claim 13 or 14, wherein the coating comprises a nanomaterial.

16. The method of claim 15, wherein the nanomaterial comprises carbon.

17. The method of claim 15, wherein the nanomaterial comprises gold.

18. The method of claim 15, wherein the nanomaterial comprises zinc oxide.

19. An article for biosensing, comprising: an electrode; a conductive polymer disposed on a surface of the electrode; and a biomolecule covalently bound to the conductive polymer, wherein the conductive polymer comprises a functional group including a nitrophenyl ring.#14581396vl20. The article of claim 19, further comprising a voltage source in electrical communication with the electrode.

21. The article fo claim 19 or 20, wherein the conductive polymer is polypyrrole, polyaniline, and / or poly(3,4-ethylenedioxythiophene).

22. The article of any one of claims 19-21, wherein the biomolecule is an antibody.

23. The article of any one of claims 19-22, further comprising an antigen bound to the antibody.#14581396vl

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